Application of the inhibitor Ciclopirox in porcine reproductive and respiratory syndrome

Abstract

The present invention provides an application of an inhibitor, ciclopirox, in porcine reproductive and respiratory syndrome. After treating cells with the specific inhibitor of eIF5A hypusination modification, ciclopirox, the proliferation of PRRSV can be inhibited. Taking PAMs, CRL-2843-CD163 cells and MARC-145 cells as examples, after treating the cells with the specific inhibitor of eIF5A hypusination modification, ciclopirox, and then inoculating with PRRSV, by detecting the expression of PRRSV N protein and the change of PRRSV titer, it can be known that the inhibitor ciclopirox has an inhibitory effect on the proliferation of PRRSV. The present invention lays a technical foundation for the prevention and control of PRRS, and also provides new ideas and references for the screening of new anti-PRRSV drugs.

Description

Technical Field

[0001] The present invention relates to the application of an inhibitor ciclopirox in porcine reproductive and respiratory syndrome, belonging to the technical field of animal disease prevention and control. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, was first discovered in North America in 1987 and has since spread widely around the world. Since it was first reported in China in 1995, the disease has been prevalent in the country for a long time and is one of the major infectious diseases affecting the pig industry in China. PRRS is a viral infectious disease mainly characterized by abortion in sows and respiratory disorders in pigs of all ages, caused by porcine reproductive and respiratory syndrome virus (PRRSV). PRRSV is a single-stranded positive-sense RNA virus, belonging to the order Nidovirales, the family Arteriviridae, and the genus Arteriviridae, with a full-length genome of 15.4 kb. PRRS has the following characteristics that make prevention and control difficult: first, there is persistent infection; second, the virus highly mutates; third, antibodies cannot produce effective immune protection. Due to the relatively fast mutation of PRRSV and the presence of multiple genotypes and sub-genotypes, the effect of immunoprevention and control using vaccines is not ideal. The current prevention and control measures mostly focus on aspects such as disinfection of the pig farm environment and improvement of the immunity of live pigs. Therefore, it is an extremely urgent scientific research task to specifically develop relevant prevention, control and treatment drugs.

[0003] Eukaryotic translation initiation factors (eIFs) are a family of proteins that play important roles in the process of eukaryotic protein translation. A large number of studies have shown that viruses can inhibit the recruitment of host mRNA by ribosomes and create an environment conducive to their own replication by regulating the expression and modification of eIFs. Existing studies have confirmed that eukaryotic translation initiation factor 5A (eIF5A) is closely related to virus replication. The Rev protein of the Human Immunodeficiency Virus (HIV) can bind to eIF5A and continuously shuttle between the nucleus and cytoplasm of host cells, mediating the nuclear-cytoplasmic transport of incompletely spliced viral mRNA. The loss-of-function mutant of eIF5A blocks the nuclear export of the Rev protein and the replication of HIV. Further studies using the yeast two-hybrid technique found that the Rev protein of HIV forms a complex with ribosomal protein L5 through eIF5A to obtain the nuclear export pathway of mRNA. Treating cells with an antibody against ribosomal protein L5 will cause the viral mRNA to fail to be normally transported to the cytoplasm and the viral structural proteins to fail to be normally expressed.

[0004] eIF5A is currently the only known protein with hydroxyproline modification. The hydroxyproline residue is an important group for eIF5A to exert its biological functions and also plays an important role in the process of virus replication. Since the hydroxyproline modification of eIF5A involves two enzyme-catalyzed steps of strict deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH), the compound Ciclopirox (CPX), whose Chinese name is ciclopirox olamine, can inhibit the activity of DOHH, thereby inhibiting the formation of eIF5A hydroxyproline modification. Treating cells with a hydroxyproline modification inhibitor results in the inability of HIV to replicate and a 30% decrease in the Ebola virus titer. These results suggest that the hydroxyproline modification of eIF5A is a novel antiviral drug target. PRRSV and HIV are both RNA viruses. However, whether Ciclopirox will affect the replication of PRRSV has not been reported yet. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide an application of the inhibitor Ciclopirox in porcine reproductive and respiratory syndrome.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] Application of the inhibitor ciclopirox in porcine reproductive and respiratory syndrome.

[0008] The inhibitor ciclopirox is a specific inhibitor of eIF5A hydroxylation modification.

[0009] Application of the inhibitor ciclopirox in the preparation of a drug for inhibiting the proliferation of porcine reproductive and respiratory syndrome virus.

[0010] Among them, the concentration of the inhibitor ciclopirox for treating cells is 5-10 mmol / L;

[0011] Preferably, the concentration of the inhibitor ciclopirox for treating cells is 10 mmol / L.

[0012] Advantages of the present invention:

[0013] The present invention relates to an application of an inhibitor ciclopirox in porcine reproductive and respiratory syndrome. After treating cells with the specific inhibitor ciclopirox of eIF5A hydroxylation modification, the proliferation of PRRSV can be inhibited. Taking MARC-145 cells (African green monkey embryonic kidney cells), porcine alveolar macrophages (PAMs) and the porcine alveolar macrophage cell line CRL-2843-CD163 stably expressing the CD163 receptor as examples, after treating cells with the specific inhibitor ciclopirox of eIF5A hydroxylation modification and then inoculating PRRSV, by detecting the expression of PRRSV N protein and the change of PRRSV titer, it can be known that the inhibitor ciclopirox has an inhibitory effect on the proliferation of PRRSV. Based on this result, this inhibitor lays a technical foundation for the prevention and control of PRRS, and also provides new ideas and references for the screening of new anti-PRRSV drugs. Description of the drawings

[0014] Figure 1 Effect of different concentrations of the inhibitor ciclopirox on cell viability.

[0015] Figure 2 Effect of treating cells with Ciclopirox (CPX) on PRRSV titer.

[0016] Figure 3 Effect of treating cells with Ciclopirox (CPX) on PRRSV proliferation detected by IFA.

[0017] Figure 4 Verification of the effect of treating cells with Ciclopirox (CPX) on the expression of PRRSV N protein by Western Blot. Specific Embodiments

[0018] The following further details the specific embodiments of the present invention in conjunction with the examples. Unless otherwise specified, the instruments and equipment involved in the examples are all conventional instruments and equipment; the reagents involved are all commercially available conventional reagents; the test methods involved are all conventional methods.

[0019] Biological materials involved:

[0020] HN07-1 strain is a highly pathogenic strain of PRRSV, isolated and identified by the Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences (GeneBank accession number: KX766378.1) and generously provided; MARC-145 cells, a commonly used experimental cell, can be obtained from public channels. PAMs were collected from 4- to 6-week-old specific pathogen-free piglets (negative for antigen and antibody detection of PRRSV, PCV2, PRV, CSFV, PPV, etc.). CRL-2843-CD163 cells are a porcine alveolar macrophage cell line stably expressing the CD163 receptor, generously provided by Northwest A&F University. The synthesis and sequencing of related primers were provided and completed by Bioengineering (Shanghai) Co., Ltd.

[0021] Experimental reagents involved:

[0022] The eIF5A hypusination-specific inhibitor Ciclopirox was purchased from MCE. DMEM, RPMI1640 medium, EDTA-trypsin, DEPC water, urea, Trisbase, RIPA lysis buffer were purchased from Solarbio Science & Technology Co., Ltd., fetal bovine serum was purchased from Hangzhou Sijiqing Company, PVDF membrane, DAPI (4,6-diamidino-2-phenylindole-dihydrochloride) dye and TRIZOL reagent were purchased from Shanghai Beyotime Biotechnology Co., Ltd., the reverse transcription kit was purchased from TAKARA, the fluorescence quantitative kit was purchased from Roche, and the Cell Proliferation Assay detection kit was purchased from Promega. The monoclonal antibody against PRRSV N protein was prepared and stored in our laboratory, and the β-Actin monoclonal antibody was purchased from CST.

[0023] Example 1 Effects of Different Concentrations of the Inhibitor Ciclopirox on Cell Viability

[0024] (1) Prepare the virus

[0025] The PRRSV HN07-1 strain with an MOI of 1 was inoculated into MARC-145 cells that had grown into a monolayer, and a maintenance medium with a serum content of 3% was added. The cells were then placed in an incubator with a 5% CO2 content. When more than 80% of the cells showed cytopathic effects, they were repeatedly frozen and thawed multiple times to completely break the cells and fully release the virus. The mixture was centrifuged at 12,000 rpm for 10 min, and the supernatant was filtered through a 0.22-μm microporous membrane. The virus was aliquoted at 500 μL / tube and stored at -80 °C for later use.

[0026] (2) Effect of the inhibitor Ciclopirox on cell viability

[0027] CRL-2843-CD163 cells were cultured in a 96-well cell culture plate. When they covered more than 80% of the bottom area, Ciclopirox at concentrations of 1, 2, 5, 10, 20, 50, and 100 mM was added to the wells, with 8 replicates for each concentration. The wells with the medium added (Ciclopirox concentration of 0 mM) were used as the control. After adding the inhibitor Ciclopirox, the cell culture plate was placed back into the CO2 incubator and incubated for another 30 min before being taken out. The absorbance at a wavelength of 490 nm was measured using a spectrophotometer. The results are as Figure 1 shown. Compared with the control group, a Ciclopirox concentration of 10 mM had the least effect on the cell growth state among the maximum doses, so this concentration was selected for subsequent experiments.

[0028] Example 2 Effect of the inhibitor Ciclopirox on the PRRSV titer

[0029] The median tissue culture infective dose (TCID 50 ) method was used to determine the change in the PRRSV HN07-1 titer after treating cells with the inhibitor Ciclopirox. The specific procedure is as follows:

[0030] (a) PAMs and CRL-2843-CD163 cells treated with 10 mM inhibitor Ciclopirox and untreated were inoculated with PRRSV HN07-1 with an MOI of 1. Untreated PAMs and CRL-2843-CD163 cells were used as the control. Cell samples were harvested at 36 hpi (hours post infection, hpi), repeatedly frozen and thawed 3 times, and centrifuged at 12,000 rpm to remove cell debris. The viruses were harvested separately.

[0031] (b) First, wash the MARC-145 cells covering 80% of the bottom wall 3 times with PBS. After trypsin digestion, disperse them into single cells and add them to a 96-well plate using a multichannel pipette. When the cells grow to cover 80% of the bottom area of the well, conduct the subsequent virus inoculation experiment;

[0032] Adjust the PRRSV HN07-1 harvested in step (a) to 10 -1 to 10 -10 a total of 10 dilution gradients, and inoculate 100 μL / well onto the confluent monolayer MARC-145 cells respectively. Inoculate 8 wells for each dilution gradient, and use the MARC-145 cells not infected with the virus as a control. Continue to culture in a CO2 incubator for 5 days; Record the cytopathic effects of each well, and repeat the experiment 3 times. Calculate the TCID 50 value according to the Reed-Muench method.

[0033] Treat PAMs and CRL-2843-CD163 cells with the inhibitor Ciclopirox at a concentration of 10 mM and then inoculate PRRSV HN07-1 (Ciclopirox remains in the medium all the time). Harvest the virus at 36 hpi and compare it with the PAMs and CRL-2843-CD163 cells that were only inoculated with PRRSV HN07-1 without adding the inhibitor Ciclopirox. The measured TCID 50 value results are as Figure 2 . The TCID 50 experiment confirmed that the titer of PRRSV HN07-1 decreased significantly after treating PAMs and CRL-2843-CD163 cells with the inhibitor Ciclopirox (P < 0.05).

[0034] Example 3 Detection of virus proliferation by IFA method

[0035] After treating PAMs with Ciclopirox at a concentration of 10 mM, infect the PAMs with PRRSV HN07-1 at an MOI of 0.1. Use DMEM medium containing 3% serum (Ciclopirox remains in the medium all the time). At the same time, set the PAMs that were only infected with PRRSV HN07-1 without adding the inhibitor Ciclopirox as a control. Discard the medium at 24 hpi, gently wash the cells 3 times with pre-cooled PBS, fix the cells with methanol for 30 min, use 5% skim milk prepared with PBST to block the cells at room temperature for 1 h, wash the cells 3 times with PBST, incubate the cells with anti-N protein monoclonal antibody at 37 °C for 1 h, wash the cells 3 times again with PBST, incubate with CY3-labeled goat anti-mouse secondary antibody at 37 °C in the dark for 1 h, and then detect the fluorescence signal of the cells. Stain the cell nuclei with DAPI dye, and take fluorescence pictures with a laser confocal Olympus FLUOVEIW IX81.

[0036] IFA test confirmed that after treating cells with the inhibitor Ciclopirox and then inoculating with PRRSV HN07-1, compared with PAMs that were only inoculated with PRRSV HN07-1 without adding the inhibitor Ciclopirox, the fluorescence signal in the Ciclopirox treatment group was significantly weakened, indicating that the replication of PRRSV HN07-1 was significantly inhibited( Figure 3 ).

[0037] Example 4 Western Blot verification of the effect of treating cells with the inhibitor Ciclopirox on PRRSV replication

[0038] Western Blot detection: After treating PAMs with the inhibitor Ciclopirox at a concentration of 10 mM and then inoculating with the PRRSV HN07-1 strain with an MOI of 0.1 (Ciclopirox was present in the medium all the time), at the same time, PAMs that were only infected with PRRSV HN07-1 without adding the inhibitor Ciclopirox were used as a control. At 24 hpi, cells from both groups were harvested. The cells were washed 3 times with PBS, digested and harvested with EDTA-Tripsin, lysed on ice with RIPA lysis buffer for 30 min, the proteins were transferred to a PVDF membrane after SDS-PAGE detection, the PVDF membrane was blocked with 5% skim milk prepared with PBST at room temperature for 2 h, incubated with a monoclonal antibody against PRRSV N protein and a monoclonal antibody against β-Actin at 37 °C for 1 h respectively, the PVDF membrane was washed 3 times with PBST, then incubated with HRP-labeled goat anti-rabbit IgG for 1 h, washed 3 times with PBST, and the signal was detected with ECL hypersensitive luminescent solution. Western Blot detected that the expression level of PRRSV N protein in the Ciclopirox treatment group was significantly lower than that in the untreated group( Figure 4 ), indicating that the inhibitor Ciclopirox can inhibit the replication of PRRSV.

Claims

1. Use of the inhibitor ciclopirox in the preparation of a drug for inhibiting the proliferation of porcine reproductive and respiratory syndrome virus.

2. The application according to claim 1, wherein The inhibitor ciclopirox is a specific inhibitor of eIF5A hydroxylation modification.

3. The application according to claim 1, characterized in that, When the inhibitor ciclopirox is used to treat cells, the concentration is 5 - 10 mmol / L.

4. The application according to claim 3, characterized in that, When the inhibitor ciclopirox is used to treat cells, the concentration is 10 mmol / L.

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